To diagnose a disease, a physician often looks for the appearance of a chemical marker in a patient. The chemical marker is a specific compound that is expressed in either abnormally high or low amounts during the course of an illness. A clinician examines a patient's plasma and/or cerebrospinal fluid using an assay specifically designed to quantitatively determine the concentration of the marker. A physician observing unusual concentrations of the marker understands that the patient may be suffering from an associated illness.
L-lactate is a chemical marker that is associated with a number of pathological states which result from a reduced oxygenation of biological fluids. Shock, pneumonia and congestive heart failure produce increased levels of L-lactate in plasma. One observes abnormally high cerebrospinal fluid levels of L-lactate as the result of bacterial meningitis, hypocapnia and cerebral ischemia.
Scientists have developed a number of enzymatic assays for the quantitative determination of L-lactate in biological solutions. The most commonly used assay is based on the enzymatic conversion of lactate to pyruvate (Scheme 1). See U.S. Pat. No. 4,166,763. L-lactate is oxidized to pyruvate by lactate oxidase (LOD). The resulting hydrogen peroxide is utilized by peroxidase (POD), which induces the coupling of a hydrogen donor and a coupling agent; a colored dye (chromogen) is formed. The concentration of the chromogen is measured spectrophotometrically. Because the concentration of chromogen is directly proportional to the concentration of lactate in the initial solution, one can calculate an observed lactate concentration. ##EQU1##
Clinical L-lactate assays are primarily performed by technicians in hospital laboratories. These assays are usually not run as part of a routine panel of tests. Where a patient is suffering from a reduced oxygenation of biological fluids, there is a critical need for an immediate diagnosis. Accordingly, a technician must be able determine L-lactate concentrations as quickly and efficiently as possible.
There are several steps that a technician takes to run an enzyme based, spectrophotometric assay for L-lactate. First a proper wavelength for the spectrophotometric measurement of the chromogen is chosen. Second, a "calibration factor" is established that allows the mathematical conversion of the spectrophotometric measurement of the chromogen to an observed L-lactate concentration. Plasma or cerebrospinal fluid sample from a patient is collected and prepared for testing. Finally, the prepared sample is mixed with the proper enzymatic reagents and there is a spectrophotometric measurement of the absorbence of radiation by the chromogen.
The technician establishes the calibration factor for L-lactate concentration by correlating the original concentration of L-lactate in a sample with the amount of radiation absorbed by the resulting chromogen. To establish this correlation, one has to be able to separate out the absorbence of the chromogen from the absorbence of other components in the assay system. This process can be represented by Scheme 2. ##EQU2##
The technician combines known concentrations of the assay reagents: lactate oxidase, peroxidase, a hydrogen donor, a coupling agent, and any other chemical element needed to perform the spectrophotometric determination. An aliquot is removed from the reagent combination and absorbence is read on a spectrophotometer. To the stock reagent solution, the technician adds a predetermined amount of L-lactate and allows the enzymatic reactions providing the chromogen to proceed until they are essentially complete. An aliquot of the chromogen containing solution is then removed and a second absorbence reading on the spectrophotometer is taken. The difference between the technician's final and initial spectrophotometric readings is the "calibration factor."
A technician can employ a single set of reagents over an extended period of time; 90 days is not unusual where the technician refrigerates the open containers. Because the reagents are not stable, the calibration factor must be determined frequently during this time period. If one could calculate the calibration factor for a reagent set once, and then use that number for every spectrophotometric assay run with the reagent set, a substantial amount of time in performing an L-lactate assay would be saved. This time savings would allow a physician to more quickly and efficiently interpret the assay results and render a diagnosis.